Suppression of immunostimulatory siRNA-driven innate immune activation by 2′-modified RNAs

Abstract

Single-stranded (ss) and double-stranded (ds) small interfering RNAs (siRNAs) containing immunostimulatory RNA motifs can activate innate immunity through Toll-like receptor 7/8 (TLR7/8), leading to the production of proinflammatory cytokines and type I interferon. More recently, we have noted that 2′-uridine modified ss or ds siRNAs not only evade immune activation, but can suppress TLR signaling triggered by their unmodified counterparts. Here we compared the inhibitory effects of several 2′-modifications. In contrast to 2′-deoxy uridine modified ss siRNAs, 2′- O-methyl uridine modified ss siRNAs inhibited at nanomolar concentrations the production of TNF-α induced by a variety of immunostimulatory RNA sequences. Using oligonucleotide microarrays, we highlight the strong suppressive effect of RNA-containing 2′- O-methyl uridines. Indeed, nearly all of the 270 genes induced by an immunostimulatory ss siRNA were completely inhibited or downregulated by cotreatment with its 2′- O-methyl modified version. Also, 2′- O-methyl modified RNAs inhibited E. coli total RNA or mitochondrial RNA to induce TNF-α production in human monocytes. Collectively, these data indicate that 2′-modified RNAs, in particular those containing 2′- O-methyl modification, are recognized with high affinity by TLR7/8, but do not induce downstream signaling. Therefore, this new generation of TLR antagonists can be used as immunosuppressive agents to interfere with TLR signaling.